Forward chemical genetics is an emerging field that offers powerful tools to search for novel drug candidates and their targets (Lokey, R. S., “Forward Chemical Genetics: Progress and Obstacles on the Path to a New Pharmacopoeia,” Curr Opin Chem Biol 7:91-6 (2003); Tan, D. S., “Sweet Surrender to Chemical Genetics,” Nature Biotech. 20:561-563 (2002); Specht et al., “The Emerging Power of Chemical Genetics,” Current Opinion in Cell Biology 14:155-159 (2002); and Schreiber, S. L., “The Small Molecule Approach to Biology,” Chem Eng News, pp. 51-61 (2003)). It differs from classical genetics by substituting small molecules for mutation inducing agents or X-ray irradiation. Using combinatorial techniques, (Jung, G., “Combinatorial Chemistry: Synthesis, Analysis, Screening,” Wiley-VCH, Weinheim: Cambridge (1999); Nicolaou et al., “Handbook of Combinatorial Chemistry: Drugs, Catalysts, Materials,” Wiley-VCH, Weinheim (2002)) one is able to rapidly screen a large number of small molecules and identify those that induce a novel phenotype in a cellular or embryonic system. Once a phenotypic effect is found, the next step is to identify the biological target using an affinity matrix made of the immobilized hit compound. However, the synthesis of an efficient affinity matrix without loss of activity by the hit compound has been shown to be challenging, or sometimes totally impossible due to the difficulties of adequate linker attachment.
Insulin or insulin-like growth factors (IGF) play essential roles in growth, development, and the maintenance of normal metabolic homeostasis including glucose uptake from the blood stream (Le Roith et al., “Recent Advances in Our Understanding of Insulin Action and Insulin Resistance,” Diabetes Care 24:588-597 (2001)). These signaling pathways are closely related to diabetic disease/obesity/aging processes and are highly conserved from yeast to humans, but the biochemical mechanism is not yet fully understood. A small molecule regulator for insulin/IGF downstream of the target protein will provide a useful tool and information to dissect the signaling mechanism.
In C. elegans, the Daf-2 (36% identical to the insulin receptor and 35% identical to the IGF-1 of human; there is no known insulin receptor in C. elegans (Le Roith et al., “Recent Advances in Our Understanding of Insulin Action and Insulin Resistance,” Diabetes Care 24:588-597 (2001)) signaling pathway controls food intake, metabolism, growth and life span, and shares, at least partially, common down-stream players with mammalian cells, including phosphoinositide-3 kinase (PI3K). PI3K catalyzes the conversion of phosphatidyl inositol[4,5]bisphosphate (PIP2) into the active signaling molecule, phosphatidyl inositol[3,4,5]trisphosphate (PIP3), and one of the important end results in mammalian cells, is glucose uptake into the cell via protein kinase B (Akt) activation (Hawkins et al., “Platelet-Derived Growth-Factor Stimulates Synthesis of Ptdlns(3,4,5)P3 by Activating a Ptdlns(4,5)P2 3-Oh Kinase,” Nature 358:157-159 (1992)). A single mutation of a kinase domain of Daf-2 (i.e., e1370), which causes constitutive arrest at the dauer larval stage (at the restrictive temperature 25° C., whereas normal phenotype occurs at the permissive temperature of 16° C.), was chosen for this study (Gems et al., “Two Pleiotropic Classes of Daf-2 Mutation Affect Larval Arrest, Adult Behavior, Reproduction and Longevity in Caenorhabditis elegans,” Genetics 150:129-155 (1998)). Dauer larvae have slowed metabolic rates, store large amounts of fat, and live longer than reproductive adults. The uniquely small size phenotype compared to a normal adult worm serves as a simple readout for the primary screening (Carroll et al., “Model Systems in Drug Discovery: Chemical Genetics Meets Genomics,” Pharmacol Ther 99:183-220 (2003); Choy et al., “Fluoxetine-Resistant Mutants in C. elegans Define a Novel Family of Transmembrane Proteins,” Mol Cell 4:143-52 (1999)).
The present invention overcomes the problem of inefficient affinity matrix synthesis and allows for the isolation of hydrophobically-capped bioactive compounds for use in treating patients with diabetes and obesity.